AbstractArsenic is a ubiquitous environmental toxicant with antimicrobial properties that can be found in food and drinking water. The influence of arsenic exposure on the composition of the human microbiome in US populations remains unknown, particularly during the vulnerable infant period. We investigated the relationship between arsenic exposure and gut microbiome composition in 204 infants prospectively followed as part of the New Hampshire Birth Cohort Study. Infant urine was analyzed for total arsenic concentration using inductively coupled plasma mass spectrometry. Stool microbiome composition was determined using sequencing of the bacterial 16S rRNA gene. Infant urinary arsenic related to gut microbiome composition at 6 weeks of life (p = 0.05, adjusted for infant feeding type and urine specific gravity). Eight genera, six within the phylum Firmicutes, were enriched with higher arsenic exposure. Fifteen genera were negatively associated with urinary arsenic concentration, including Bacteroides and Bifidobacterium. Upon stratification by both sex and feeding method, we found detectable associations among formula-fed males (p = 0.008), but not other groups (p > 0.05 for formula-fed females and for breastfed males and females). Our findings from a US population indicate that even moderate arsenic exposure may have meaningful, sex-specific effects on the gut microbiome during a critical window of infant development.